University: Gent
Faculty: Sciences
Department: Biology
Unit: Nematology
Academic year: 2015-2016
Potential of Paenibacillus spp. as bio control agent
for root-knot nematodes (Meloidogyne spp.)
Jackline A. Bakengesa
Promoter & Supervisor: Prof. Dr. Ir. Wim Wesemael
Thesis submitted as to obtain the degree of Master of Science in Nematology
Potential of Paenibacillus spp. as bio control agent for rootknot nematodes (Meloidogyne spp.)
Jackline A. BAKENGESA1, Wim M. L. WESEMAEL1,2,3
1
Department of Biology, Faculty of Sciences, Ghent University, K.L Ledeganckstraat 35, B-9000, Ghent,
Belgium
2
Institute for Agricultural and Fisheries Research (ILVO), Burgemeester Van Gansberghelaan 96, 9820
Merelbeke, Belgium
3
Laboratory for Agrozoology, Faculty of Bioscience engineering, Ghent University, Coupure Links 653,
B-9000 Ghent, Belgium
Bakengesa J & Wesemael W
Abstract; Root-knot nematodes are the most damaging plant-parasitic nematodes. Potential of
Paenibacillus spp. as a bio-control agent for root-knot nematodes (M. fallax, M. chitwoodi, M.
javanica, M. enterolobii, M. incognita and M. hapla) was examined in vitro and in vivo. This
includes its effect on nematode attraction, penetration, mortality, hatching and multiplication.
Plant growth and phytotoxic effect were also assessed. All in vitro experiments were done at
room temperature. Plants in pot experiment were grown in the growth chamber (16 hours light, 8
hours dark with temperature between 22°C and 24°C). 10% and 100% bacterial suspension (BS)
were used as treatments with buffer and distilled water as control. 100% Paenibacillus spp. BS
strongly caused J2 mortality and reduced hatching (p<0.05). Hatching was inhibited by 97%99% for all tested species. More than 90% J2 mortality occurred after 24 hours of exposure in
100% BS. Some J2 recovered when exposed in distilled water with increasing time. 10% BS also
reduced final population of all tested species of root-knot nematodes in a pot experiment. Root
gall index was the same for all treatments. Phytotoxic effect on plants was observed at 100% BS
but seems to be influenced by climatic conditions particularly temperature. Increase of plant
growth due to Paenibacillus spp. was also observed. Paenibacillus spp. was found to have
nematistat and nematicidal effects on root-knot nematodes.
Key words; Biological control, mechanism, Paenibacillus spp., root-knot nematodes
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Bakengesa J & Wesemael W
Plant-parasitic nematodes cause a loss of about $125 billion worldwide due to annual
crop losses (Chitwood, 2003). Among all, root-knot nematodes have increased in importance in
different parts of the world including Europe and Africa (Wesemael et al., 2011; Onkendi et al.,
2014) and cause substantial damage. Different species of root-knot nematodes have been found
from warmer to cooler areas. For example the more aggressive species M. enterolobii has been
found in Europe and different parts of Africa such as DRC, Burkina Faso, Malawi, South Africa
Mozambique, Togo, and Senegal.
Root-knot nematodes, (genus Meloidogyne Goldi, 1887) penetrate within plant roots,
feed, reproduce and induce small to large galls triggering a plant disease known as root-knot.
They are of great economic importance as they disrupt plant physiology and may reduce crop
yield and quality due to their endoparasitic form of living and feeding (Karssen et al., 2013).
Chemical control has been worldwide used as a primary means of control, not only for
root-knot nematodes but also for other plant pathogens as well. These chemicals have several
negative impacts to the environment, biodiversity and humans at large which led to a total
prohibition or restricted use of some of the nematicides (Nyczepir & Thomas, 2009; Haydock et
al., 2013). Hence causes the impetus for new, urgent, safe and more effective approaches. This is
necessary to ensure higher agriculture production to fit the growing world population while
keeping the environment safe for present and future generations.
To fulfill this goal organic inputs and microbial inoculants have become main interests
(Chauhan et al., 2015). This is due to the fact that these could be the best alternative for the
chemical products for sustainable agriculture (Ashraf et al., 2013) such as the use of biocontrol
agents. Promising results have been obtained and repeatedly tested on control of plant-parasitic
nematodes by using antagonistic bacteria (Giannakou et al., 2004) such as Pseudomonas
aeruginosa (Siddiqui et al., 2000) on control of Meloidogyne spp. Rhizobacteria assists on
triggering plants endogenous defense mechanisms i.e. Pseudomonas fluorescens (M'piga et al.,
1997). Root occupation by rhizosphere bacteria also lessen nematode invasion (Siddiqui &
Shaukat, 2004). Xiong et al. (2015) showed the efficacy of bacterium Bacillus firmus YBf-10 as
a biocontrol agent. It exhibits nematicidal activity against root-knot nematodes including
motility, inhibition of hatching and above all lethal activity. Nematicidal effect is due to activity
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Bakengesa J & Wesemael W
of bioactive secondary metabolites. Its efficacy is comparable with that of fenamiphos, the
broadly used chemical nematicide.
Biological control basically involves the usage of microbial agents (living organisms) for
management of plant diseases and pests (Karssen et al., 2013). Bio-control can be geared by
some organisms including fungi, mites and bacteria. The study by Hashem and Abo-Elyousr
(2011) reported significant results due to application of different biocontrol agents such as;
mortality of nematodes, induction of systemic resistance in plants and significant growth due to
availability of enough nutrients. Among others, one of the extensive studied biological control
agents are plant growth promoting rhizobacteria (PGPR) (Khan et al., 2008). PGPR have
beneficial effects on seed germination, emergence and colonization of roots, mineral nutrition
and water utilization, suppression of diseases and hence overall plant growth (Siddiqui et al.,
2007).
PGPR operation mechanisms is either directly, indirectly or combination of both
(Martínez-Viveros et al., 2010; Chauhan et al., 2015). Direct mechanisms include assisting on
uptake of essential nutrients and secretion of plant growth promoting metabolites like cytokinins,
indole acetic acid (IAA), gibberellins, etc., Indirect mechanism is through production of
antibiotics to reduce or prevent pathogenic effects, such compounds are siderophores, hydrogen
cyanide (HCN), etc. Within PGPRs some have reached the stage of commercial success such as
Azospirillium and Bacillus while others not yet as Paenibacillus (Ashraf et al., 2013).
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Bakengesa J & Wesemael W
Figure 1: The important benchmark of novel PGPR that make them great biocontrol agents for
application and future commercialization (Chauhan et al., 2015).
Regardless reasonable public and legislative pressure to lessen the use of nematicides due
to possible health and environmental risks, only a minority of biocontrol agents have been
established and none is widespread in use (Viaene et al., 2013). But researches are done
worldwide on different organisms for their possibility to be used on control, a rhizobacteria
Paenibacillus being one.
Paenibacillus spp. is among PGPR that forms a biofilm around roots (root tips). In this
respect it can hamper penetration of harmful organisms to plants particularly root-knot
nematodes hence the potential to be used as biocontrol agent. Timmusk et al. (2005)
recommended P. polymyxa as potential biocontrol agent for commercial purpose due to its ability
to form endospores, production of several kinds of antibiotics and possibility to colonize several
host plants. Khan et al. (2008) found exposure of root-knot nematode M. incognita to various
concentrations of culture filtrate of P. polymyxa GBR-1 under in vitro conditions significantly
reduced hatching from eggs and caused substantial mortality of its juveniles.
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Bakengesa J & Wesemael W
Studies on P. polymyxa as a potential tool for bio control mostly has been based on
antibiotics production (Timmusk et al., 2005). Its mechanism of action on nematode reduction
was suggested area of study by Khan et al. (2008). In this study, the potential of Paenibacillus
spp. and its mechanism of action as a biocontrol agent for Meloidogyne spp. were assessed on
concentration basis. The experiments were conducted under in vitro and greenhouse conditions.
The effects of Paenibacillus spp. on nematode attraction, penetration, mortality, hatching and
multiplication were examined and reported.
MATERIALS AND METHODS
Nematode culture
Species of root-knot nematodes (M. fallax, M. chitwoodi, M. javanica, M. enterolobii, M.
incognita and M. hapla) were obtained from pure cultures maintained at ILVO, Merelbeke,
Belgium. Cultures were multiplied on tomato and/or in transparent closed containers on potato
tubers.
Multiplication on tomato plants, cultivar Marmande was used. Seedlings were allowed to
develop a dense root system in organic soil (PeltrAcom N.V, NPK fertilizer added (14-16-18)
1.4kg/m3, PH 5.8). After 4 weeks they were transplanted in 2l pots filled with heat sterilized soil
(100°C, 16h). Then 2000 of pure culture root-knot nematode juveniles (J2) were inoculated.
Each day the plants were watered to field capacity. Nematodes were left to multiply for three
months and then harvested.
In closed containers, potato tubers (cultivar Bintje) were used. Tubers were cleaned with
tap water and then submerged in a 5% sodium hypochlorite (NaOCl) solution and left for four
minutes to disinfect the tubers. The potatoes were then rinsed with tap water to remove residual
NaOCl. Potato tubers were dried and left to sprout for three weeks.
Transparent closed containers (11.5cm diameter and 8.5cm length) were filled with 200g
of sterilized white sand soil and watered with 30ml of sterile tap water. Per container one
sprouted potato tuber was placed with the point of sprout touching the sand. These closed
containers with potato tubers were then stored in the dark room at 20°C until formation of roots.
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Then 2000 freshly hatched J2 of root-knot nematodes were inoculated into each container and
left to reproduce. After two months the nematodes were ready to be harvested.
Nematode inoculums were prepared from maintained pure cultures. Infected tomato and
potato plants were uprooted and the entire root system gently cleaned with water to remove the
soil. The roots were then cut into small pieces of about 2mm and placed on Baermann funnel
(Baermann, 1917). Freshly hatched J2 were collected after every 24 hours. After each collection
the water in the Baermann funnel was refreshed. The collected nematode suspension was then
thoroughly homogenized and a subsample of 5ml was poured on a counting dish. With the aid of
a microscope and a counter, the nematodes were counted three times in 5ml aliquots to calculate
the nematode density. The inoculum was used for both in vitro and pot experiments.
Bacteria culture
Paenibacillus spp. was grown in Brain Heart Infusion (BHI) (OXOID LTD, Basingstoke,
and Hampshire, England) agar plates and liquid medium.
For BHI agar plates, 18.5g of BHI was mixed with 7.5g of bacterial agar and dissolved in 475ml
of distilled water. The mixture was shaken to dissolve and autoclaved at 121°C for 15 minutes.
25ml of sterilized 10% glycerol was added, shaken thoroughly, then poured on agar plates and
left to cool and solidify under laminar flow. The plates were stored in the refrigerator at 4°C.
Liquid medium was prepared by dissolving 18.5g of BHI medium in 475ml of distilled
water. The mixture was autoclaved at 121°C for 15 minutes. 25ml of sterilized 10% glycerol was
added and shaken thoroughly. The medium was then stored at room temperature.
Paenibacillus spp. culture was obtained from bacteriology department –ILVO. A single
bacterial colony was isolated and transferred to BHI agar plates and incubated overnight at 28°C.
This was repeated twice in order to obtain pure fresh cultures. Then bacteria agar plates were
maintained at 4°C in the refrigerator. Bacteria were continuously cultured whenever required in
order to obtain fresh bacteria.
To obtain inoculum, the Paenibacillus spp. single colony was transferred into 3ml BHI
liquid medium as pre-culture and incubated overnight in an incubator shaker at 28°C and
continuously shaken at 200rpm. 1.5ml of pre culture bacteria was poured into 500ml liquid
medium as the main culture. This was also incubated overnight at 28°C and 200rpm.
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The Paenibacillus spp. suspension from the main culture was transferred to the sterilized
centrifuge bottles 175ml each. The suspension was centrifuged at 4°C for 15 minutes at 4000rpm
to form pellet. The supernatant was discarded and 500ml buffer (sodium phosphate) solution
added. Buffer was prepared by dissolving 4.26g of sodium phosphate dibasic with 2.72g of
potassium phosphate monobasic (Sigma-Aldrich Co, USA) on a magnetic hot plate to make one
liter of 0.05M buffer. This was filtered into sterilized bottles through a plastic sterilized
disposable filter (250ml volume) under vacuum. The filter has a nozzle where the tubing from
the vacuum outlet is connected to. The vacuum was turned on and the buffer poured slowly
through the filter.
The Paenibacillus spp. pellets and buffer solution were gently mixed and this was taken
as stock solution. Dilutions were made with buffer to obtain different concentrations (10% and
100%). The use of buffer was important to remove nutrients agar and medium which could have
influence on bacteria and plant during experiments. All the activities were done under laminar
flow.
Paenibacillus spp. density from the stock solution was determined by optical density and
serial dilution. Optical density was done with a spectrophotometer. 1ml of the solutions, BHI
liquid medium and buffer as blank and Paenibacillus spp. stock solution in different transparent
tubes (cuvette) were placed in the machine. Beam light was allowed to pass to determine the
density of each solution.
Colony forming units after different serial dilutions of Paenibacillus spp. stock solution
(10-1 to 10-10) were also observed. 50µl of bacterial solution after each dilution was plated on
pseudomonas agar plate. The bacteria were allowed to grow overnight in the incubator at 28°C
and the number of colonies were counted.
(Pseudomonas agar plates were prepared by dissolving 18.8g of difco pseudomonas
powder (Becton, Dickinson and Company, Sparks, USA) in half liter of distilled water; mixed
thoroughly and autoclaved at 121°C for 15 minutes. Then poured on agar plates and left to
solidify under laminar flow. Agar plates were stored in the refrigerator at 4°C until use.)
Actual Paenibacillus spp. cells were 108 Colony Forming Unit /ml, this was considered
as stock bacterial suspension (BS).
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In vitro experiment
Tomato seeds (cultivar Marmande) were grown in petri dishes (14cm diameter) with a 4
layer tissue paper moistened with distilled water. The plates were covered with parafilm and
stored in the dark room at 20°C. Seeds were left to germinate for two weeks.
Paenibacillus spp. phytotoxicity test on tomato seedlings
20 tomato seedlings were dipped into BS of concentrations 10% and 100% each for 2
hours. Distilled water and buffer were taken as control. The status of the seedlings was observed
in two experimental setups, one during a sunny day and the second during a cloudy and cool day.
The experiments were done at room temperature with average temperature 25-26°C on a sunny
day and 20-21°C on a cloudy, cool day.
Effect of Paenibacillus spp. on J2 mortality
Into tubes (10ml total volume) 5ml of different treatments (10% BS, 100% BS, buffer
and distilled water) were placed. 100 freshly hatched J2 were added and stored into dark at 20°C.
Observation of mortality was done after 3 hours and 24 hours. During observation the immobile
J2 were probed with picking needle, if not moving they were considered dead. After observation
the nematodes were transferred in distilled water to check for recovery for 3 hours and 24 hours.
Effect of Paenibacillus spp. on hatching
Infected potato and tomato roots from the stock cultures were gently cleaned. Under
microscope egg masses were picked from the root system. Three egg masses were placed on a
sieve (length 2.5cm, diameter 1cm and 48µm mesh) and placed in tubes of 10ml volume. 5ml of
distilled water and buffer as control, and Paenibacillus spp. suspension (10% and 100%) was
poured into each tube. Then tubes were covered with a perforated lid to allow oxygen exchange
but limit evaporation and stored in the dark room at 20°C. The numbers of hatched J2 were
counted weekly during four weeks. After each count, the solution was replaced according to each
treatment.
After four weeks all the solutions were replaced with distilled water. Observation on
hatching recovery was done for two weeks on egg masses previously treated with 100% BS. The
numbers of non-hatched eggs were then counted for all egg masses. Therefore egg masses were
submerged with 1% NaOCl solution and eggs separated from the gelatinous matrix with bluntly
forceps. With aid of microscope all non-hatched eggs were counted.
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Paenibacillus spp. effect on nematode attraction and penetration
Tomato seedlings were dipped into BS of different concentrations (10%, 100%), distilled
water and buffer as control for 2 hours. Seedlings were then transferred on agar plate (1% of
basic technical agar (OXOID LTD, Basingstoke, Hampshire, England) and 100 freshly hatched
J2 of RKN inoculated 1cm from the rootlet. Observation was done after 24 hours, 48 hours and 5
days. Roots of the seedling were then stained (Byrd Jr et al., 1983) to count the number of
penetrated nematodes. Roots of each plant were cut into small pieces of about 1cm and placed
into 25ml of tap water. 10ml of NaOCl solution was added into each and left for 4 minutes. This
was important to soften the roots tissues for good staining. The roots were then rinsed well with
flowing tap water. 30ml of tap water with 1ml of staining solution (3.5g acid fuschin dissolved in
250ml acetic acid and 750ml distilled water) was added into each and heated on a hot plate until
boiling point for 30 seconds. After cooling the roots were well rinsed, covered with glycerol,
heated until boiling point and left to cool. Then numbers of penetrated J2 were counted.
Liquid pluronic gel was also used. It was prepared by dissolving 287.5g of powder
pluronic gel into 800ml of distilled water in order to make one liter (Wang et al., 2009). The
mixture was then stirred with a magnetic stirrer for 24 hours in cold room (4°C) to dissolve. The
gel was then stored in the refrigerator at 4°C until use. During experiment the gel was poured in
plates to solidify at room temperature. Tomato seedlings dipped into different treatments
(bacterial suspension, buffer and distilled water) were placed on it individually. 100 freshly
hatched J2 were inoculated 1cm from the rootlet. Numbers of non-penetrated nematodes were
counted. After staining (see above section) penetrated nematodes were also counted.
Greenhouse experiments
Tomato seeds were planted in plastic pots (8.8cm diameter and length) with organic soil
and left to germinate and grow. At the fourth leaves stage the plants were shifted to the growth
chamber (16 hours light, 8 hours dark with temperature between 22°C and 24°C) and inoculated
with 10ml of 10% and 100% BS, distilled water and buffer as control. Inoculation was repeated
after two days.
After one week the plants were trans-planted in 1l plastic pots filled with heat sterilized
soil (100°C, 16h) and inoculated with 500 freshly hatched J2 of root-knot nematodes. Plants
were left to grow for eight weeks; watered at field capacity on daily basis.
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8 weeks after nematode inoculation, different data were first collected, this includes;
plant height and fruits weight. Plants were then carefully uprooted from pots and the soil
attached to the roots gently removed, root weight, gall index, fresh and dry mass of shoots were
also recorded.
Then by Automatic Zonal Centrifugation (AZC) method (Hendrickx, 1995) nematodes
were extracted from each plant whole root system as organic sample and from 200cc of
homogenized soil.
Experimental design and data analysis
All experiments were set up in a completely randomized design (factorial). Five
replications for each treatment and experiment were done for six Meloidogyne spp. (M. fallax, M.
chitwoodi, M. javanica, M. enterolobii, M. incognita and M. hapla).
Package Rstudio (R 3_2_3) was used for all statistical analyses. The assumptions for
ANOVA were tested using Shapiro test for normality and Levene’s test for homogeneity of
variances. Duncan multiple range test was performed as post-hoc test. When the assumptions
were not met, data were transformed using log(x+1) or log x. And a non-parametric KruskalWallis test was carried out.
RESULTS
In vitro experiment
Paenibacillus spp. phytotoxicity test on tomato seedlings
Observations on phytotoxic effect of Paenibacillus spp. on tomato seedlings (Table 1)
showed contradicting results on different days. On a sunny day (25-26°C), all seedlings dipped
in 100% BS became weak after 2 hours of exposure while on a cooler day (20-21°C) the
seedlings in all treatments were healthy.
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Table 1: Paenibacillus spp. phytotoxic effect on tomato seedlings at room temperature
Setup 1, on sunny
Setup 2, on a cloudy
Treatments
day (25-26°C),
day (20-21°C)
dH2O
20 healthy
20 healthy
Buffer
20 healthy
20 healthy
10%
20 healthy
20 healthy
100%
20 weak and wilt
20 healthy
Effect of Paenibacillus spp. on J2 mortality
Paenibacillus spp. BS caused significant mortality of J2 of tested species of root-knot
nematodes (P < 0.0001) compared to distilled water and buffer. This is shown in figures 2 and 3.
100% of BS caused more than 40% mortality within three hours of exposure; M. hapla and M.
chitwoodi were less killed compared to other tested species. J2 mortality caused by 10% of BS
was more pronounced after 24 hours than 3 hours. The effect of BS on J2 mortality of root-knot
nematodes increased with time of exposure. M. hapla and M. incognita seem more resistant as
after 24 hours of exposure in 10% BS, J2 mortality was less than 10% compared to M.
enterolobii, M. javanica M. fallax and M. chitwoodi with up to 60% of mortality. For both
species after 24 hours of exposure in 100% Paenibacillus spp. BS, there was more than 90% of
mortality.
Paenibacillus spp. significant effect (p-values) on J2 mortality compared to control for all
the species are: M. enterolobii p-value = 1.868e-05, M. javanica p-value = 6.986e-06, M.
incognita p-value = 1.476e-05, M. hapla p-value = 1.66e-05, M. fallax p-value = 5.79e-06, M.
chitwoodi p-value = 7.628e-06.
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Figure 2: Mortality (%) of J2 of six species of root-knot nematodes exposed to 10% and 100% of
Paenibacillus spp. BS, and distilled water and buffer as control for 3 hours. Significant
differences (p < 0.05) between treatments and species are marked with a different letter.
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Figure 3: Mortality (%) of J2 of six species of root-knot nematodes exposed to 10% and 100% of
Paenibacillus spp. BS, and distilled water and buffer as control for 24 hours. Significant
differences (p < 0.05) between treatments and species are marked with a different letter.
Observations on recovery of J2 in distilled water after treatment with 10% and 100%
Paenibacillus spp. dosage during different times are shown in Table 2. The percentage recovery
increased with time the J2 were exposed in distilled water. Total recovery (100%) was observed
for J2 treated with 10% BS during 3 hours and then exposed in distilled water for 3 hours. J2
treated with 100% BS during 3 or 24 hours showed no recovery when exposed for 3 hours in
distilled water. Exposure of J2 in distilled water for 24 hours after treatment with 10%
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Paenibacillus spp. BS resulted to more than 90% J2 recovery and less than 50% J2 recovery
after treatment with 100% BS.
Table 2: Average recovery (%) of J2 of all tested species of root-knot nematodes in distilled
water after being exposed to different concentrations of Paenibacillus spp. BS during different
times
Time exposed in Paenibacillus. spp
3hours
Species
Time for
24hours
10%BS
100%BS
10%BS
100%BS
3hours
100%
0%
0%
0%
24hours
100%
65%
90%
40%
3hours
100%
0%
0%
0%
24hours
100%
60%
81%
35%
3hours
100%
0%
0%
0%
24hours
100%
72%
99%
42%
3hours
100%
0%
0%
0%
24hours
100%
59%
98%
48%
3hours
99%
0%
0%
0%
24hours
99%
61%
85%
40%
3hours
100%
0%
0%
0%
24hours
100%
60%
90%
31%
recovery
M. enterolobii
M. javanica
M. incognita
M. hapla
M. fallax
M. chitwoodi
Effect of Paenibacillus spp. on hatching of root-knot nematodes
Paenibacillus spp. BS showed strong effect (p < 0.01) on inhibition of hatching of J2 for
all tested species of root-knot nematodes (Figure 4). Treatment with 100% of BS resulted in less
than 4% J2 hatched for the consecutive four weeks. Hatching was reduced by 97% to 99%. This
was the case for all tested species of root-knot nematodes. Hatching parameters m (the time at
which 50% of the total hatching is reached), b (the hatching rate) and c (the maximum hatching
percentage) were calculated except for 100% BS where m and b values could not be calculated
due to strong inhibition of hatching. For 10% BS, buffer and distilled water the numbers of
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hatched J2 were not significantly different. No recovery on hatching was observed in distilled
water for egg masses treated previously with 100% BS.
Figure 4: Fitted curves showing the expected cumulative (%) hatch of six species of root-knot
nematodes exposed to 10% and 100% of Paenibacillus spp. BS, and distilled water and buffer as
control for four consecutive weeks.
Due to inhibition of hatching by 100% Paenibacillus BS no hatching curves could be
calculated for this concentration and analysis was only done for the final (low) hatching
percentage (Table 3).
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Table 3: Parameters of the logistic curve y = c/(1 + exp(-b×(time-m))) describing J2 hatching of
root-knot nematodes at different concentration of Paenibacillus spp. treatment and control. The
results are the means of five replicates of the time at which 50% of the total hatching is reached
(m), the hatching rate (b) and the maximum hatching percentage (c). Significant differences
between treatments for each species are marked with a different letter.
Species
B
m
c
dH2O
Buffer
10%
dH2O
Buffer
10%
dH2O
Buffer
10%
100%
M. enterolobii
1.80a
1.66a
1.22a
0.28a
0.54a
0.25a
33.49b
66.59a
45ab
1.82c
M. javanica
1.96a
2.04a
2.58a
1.74a
2.60a
1.86a
58.87a
41.89a
51.73a
2.18b
M. incognita
1.66a
2.28a
2.28a
2.42a
2.36a
2.60a
66.65a
62.9a
61.35a
2.32b
M. hapla
1.78a
2.36a
1.78a
2.12a
2.64a
2.54a
74.01a
43.51b
35.8b
3.37c
M. fallax
1.50a
1.44a
2.18a
1.1a
1.4a
1.3a
45.76a
64.63a
50.26a
1.82b
M. chitwoodi
1.54a
1.70a
1.38a
1.60a
2.00a
1.96a
63.43a
52.95a
38.24b
0.88c
Paenibacillus spp. effect on nematode attraction and penetration
After 48 hours the number of J2 of M. enterolobii around the tomato root treated with
buffer was significantly higher compared to the one treated with Paenibacillus spp. but not with
distilled water (p = 0.04319). J2 of M. fallax around tomato root treated with distilled water was
significantly higher than the other treatments (p = 0.003766). For M. chitwoodi, M. hapla, M.
incognita and M. javanica no effect on attraction due to Paenibacillus spp. was observed after 24
hours and five days (p-value > 0.05) (results not shown).
Similarly, Paenibacillus BS had no significant effect on penetration (Figure 5) of J2 of
root-knot nematodes compared to the control (p > 0.05). On average penetration was less than
30%. For all the species tested, penetration was lower when the seedlings were treated with
100% BS but the difference was not significant (M. enterolobii p-value = 0.7895, M. javanica pvalue = 0.2605, M. incognita p-value = 0.6874, M. hapla p-value = 0.8982, M. fallax p-value =
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0.07056 and M. chitwoodi p-value = 0.7139). The average percentage penetration for each
species and treatments is shown in figure 5. Highest penetration was observed for M. hapla.
Figure 5: Penetration (%) of J2 of six species of root-knot nematodes exposed to 10% and 100%
of Paenibacillus spp. BS, and distilled water and buffer as control after five days. Significant
differences (p < 0.05) between treatments and species are marked with a different letter.
Greenhouse experiments
Paenibacillus spp. BS reduced nematode final population density (Pf) compared to buffer
and distilled water (Table 4). Paenibacillus spp. BS significantly reduced the Pf of M. fallax (P =
0.0445) compared to control. For the other tested root-knot nematodes species BS had no
significant Pf reduction (M. enterolobii p-value = 0.7417, M. javanica p-value = 0.1396, M.
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Bakengesa J & Wesemael W
incognita p-value = 0.2865, M. hapla p-value = 0.6388, M. chitwoodi p-value = 0.3409). 10% BS
rendered a lower Pf compared with the 100% BS (p-value > 0.05).
Table 4: Average Pf eight weeks after inoculation of root-knot nematodes on tomato plants
treated with 10% and 100% Paenibacillus spp. BS, buffer and distilled water as control.
Significant differences between treatments and species are marked with a different letter.
Species
Distilled water
Buffer
10% BS
100% BS
M. enterolobii
4919d
3928d
3047d
4334d
M. javanica
10910e
9393e
6326e
9567e
M. incognita
36485g
28847g
22700g
25220g
M. hapla
31749f
32951f
26042f
28083f
M. fallax
2592b
3054b
1560a
1747a
M. chitwoodi
4900c
4792c
3293c
3728c
A significant effect of Paenibacillus spp. BS on increase of plant growth was found on
plants inoculated with M. javanica where the length (p = 0.03555), shoot fresh (p = 0.000508)
and dry weight (p = 0.004822) was significantly higher (Figure 6, 7 and 8). Root weight of
tomato plants treated with bacteria and inoculated with M. incognita was also significantly
higher (p = 0.03889) compared to control (Figure 9). For plants inoculated with M. enterolobii,
the mean length, shoot fresh and dry weight was higher when treated with 100% BS (p > 0.05).
For the plants inoculated with M. chitwoodi, M. hapla and M. fallax, Paenibacillus spp.
didn’t increase plant growth parameters compared to buffer and distilled water. Plant growth
trend was also not consistent due to different treatments. Some plants treated with buffer and
distilled water showed increased growth, for example plants inoculated with M. chitwoodi,
means values for different growth parameters were higher compared to the one treated with BS
(p > 0.05).
Galling index was similar for all the plants with less than 10% galling. Tomato plants
inoculated with M. incognita, M. enterolobii and M. javanica showed more large galls and very
obvious egg masses. In all the plants flowers were present.
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Bakengesa J & Wesemael W
Figure 6: Average tomato plant length eight weeks after inoculation with six species of root-knot
nematodes and treated with 10% and 100% of Paenibacillus spp. BS, and distilled water and
buffer as control. Significant differences (p < 0.05) between treatments and species are marked
with a different letter.
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Bakengesa J & Wesemael W
Figure 7: Average tomato plant fresh shoot weight eight weeks after inoculation with six species
of root-knot nematodes and treated with 10% and 100% of Paenibacillus spp. BS, and distilled
water and buffer as control. Significant differences (p < 0.05) between treatments and species are
marked with a different letter.
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Bakengesa J & Wesemael W
Figure 8: Average tomato plant dry shoot weight eight weeks after inoculation with six species
of root-knot nematodes and treated with 10% and 100% of Paenibacillus spp. BS, and distilled
water and buffer as control. Significant differences (p < 0.05) between treatments and species are
marked with a different letter.
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Bakengesa J & Wesemael W
Figure 9: Average tomato plant root weight eight weeks after inoculation with six species of
root-knot nematodes and treated with 10% and 100% of Paenibacillus spp. BS, and distilled
water and buffer as control. Significant differences (p < 0.05) between treatments and species are
marked with a different letter.
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Bakengesa J & Wesemael W
DISCUSSION
Root-knot nematodes cause qualitative and quantitative damage in the production of
different crops. The use of chemicals as control measure is declining day to day due to negative
impact on the environment. Among alternatives different antagonistic microorganisms have been
tested to be used as bio-control agents for root-knot nematodes. Microorganisms that showed
potential are Bacillus spp. (Park et al., 2014) for M. hapla, Purpureocillium lilacinum (Kiewnick
& Sikora, 2006) and P. polymyxa (Khan et al., 2008) for M. incognita.
In this study potential of Paenibacillus spp. was studied. Preliminary observations of
phytotoxic effect of the bacteria on tomato seedlings showed that the effect can occur due to
100% BS at room temperature between 25-26°C. Higher temperature might result on increasing
phytotoxicity. Phytotoxic effect by biological control agent was also reported by Terefe et al.
(2009) on tomato seedlings where application of B. firmus at rate of 16 g/pot resulted in seedling
mortality. In this study phytotoxic effect was observed on plants on in vitro assays. Plants were
exposed direct into higher BS which is not the case for plants in the pots and field condition.
Phytotoxic effect of PGPR is related to production of IAA which is toxic at high concentration
(Lebuhn et al., 1997). Paenibacillus spp. can cause phytotoxicity on plants but seems to depend
on climatic conditions (temperature) and BS concentration. Before its application preliminary
testing can be suggested e.g. dipping of seedlings in BS.
Treatment with BS of Paenibacillus spp. in vitro showed significant effect on J2
mortality and hatching for all tested species of root-knot nematodes. Reduction of hatching and
J2 mortality occurred to a varying degree depending on the concentration of BS as compared to
control (buffer and distilled water). Mortality and immobility was observed on J2 of M.
enterolobii, M. javanica, M. incognita, M. hapla, M. fallax and M. chitwoodi treated with BS
after 3 hours and 24 hours. The effect was more pronounced with 100% BS whereby just after
three hours of exposure more than 50% of immobility occurred. Less than 10% hatching was
observed for all the species treated with 100% BS for four consecutive weeks. The reason might
be secondary metabolites and antibiotics produced by Paenibacillus spp. (Timmusk et al., 2005).
Inhibition of hatching and J2 mortality were also reported by Khan et al. (2008). P. polymyxa
GBR-1 was found to significantly inhibit hatching and caused mortality of M. incognita J2.
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Bakengesa J & Wesemael W
Observations on J2 recovery suggest that the mechanism of action of Paenibacillus spp.
can be paralysis. This implies that antibiotics and secondary metabolites found in Paenibacillus
spp. may act as nematistat and nematicidal. The study by Park et al. (2014) also reported strong
nematicidal effect of B. cereus on M. hapla. Recovery increased with time that J2 were exposed
in distilled water after the treatment. On the other hand number of recovery decreased with
increasing time of exposure into BS. The study by Jung et al. (2002) reported the antagonistic
effects to significantly increase with duration of the treatments.
Chitin degradation of nematode egg shells caused by chitinase may be a reason for
inhibition of hatching. Treatment with 100% BS completely reduced hatching and no recovery
was observed after exposure in distilled water for consecutive two weeks. This suggests that
chitinase effect on inhibition of hatching can persist for a particular amount of time. Chitinase
lysis of the nematode egg shell by Paenibacillus spp. was reported by Jung et al. (2002) where P.
illinoisensis KJA-424 caused degradation of M. incognita eggshell and resulted in the inhibition
of hatching in vitro.
Hatching of M. incognita was also completely inhibited by P. polymyxa GBR- 462, GBR508 and P. lentimorbus GBR-158 cultures (Son et al., 2009). The strains were also responsible
for increased plant growth. M. incognita hatch inhibition and J2 mortality rate was also found to
be enhanced with the increase in the concentration of BS and the crude enzymes (gelatinase and
chitinase) of P. elgii HOA73 (Jung et al., 2002).
A reduction in penetration into the seedlings treated with 100% Paenibacillus spp. (p >
0.05) may be attributed to both direct and indirect mechanism of this PGPR such as production
of secondary metabolites and root colonization. The study by Timmusk et al. (2005) showed the
possibility of P. polymyxa to form biofilms in plant roots after predominant colonization which
was not examined in this study. Biofilms can suggest inhibition of J2 penetration as they were
found to protect pathogen infection sites (Timmusk et al., 2005). Investigation on biofilm
formation is recommended for future studies to prove its possibility on protecting infection sites
that may assure failure of nematode establishment and reproduction.
Root colonization by rhizosphere bacteria was also reported by Siddiqui and Shaukat
(2004) to reduce nematode invasion. M. incognita penetration of roots, reproduction, and rootknot disease were decreased more by dual inoculations with AM fungi and PGPR (Li et al.,
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Bakengesa J & Wesemael W
2011). Production of metabolites by these rhizosphere bacteria can also reduce attraction and
penetration of nematodes as they were found to degrade specific root exudates (Oostendorp &
Sikora, 1990). Efficient rhizosphere colonization and biofilm formation was found to be one of
the important aspect for effective control (Haggag & Timmusk, 2008).
In pot experiment, some data indicated significant increase of plant growth parameters
while others did not. The increase of plant growth parameters were also reported in other studies
like Jung et al. (2002) and Khan et al. (2008). The reason for the increase may be attributed to
synthesis of plant hormones such as cytokinin and auxin (Schroth & Loper, 1986), facilitation of
nutrient availability through nitrogen and phosphate metabolism (Eastman et al., 2014). PGPR
also acts as biofertilizer (Chauhan et al., 2015). Study of PGPR P. putida, P. alcaligenes, P.
polymyxa and B. pumilus found these species to substantially increase growth of inoculated
plants (Siddiqui et al., 2007). Son et al. (2009) reported that the tested strains of Paenibacillus
species (P. polymyxa and P. lentimorbus) also promoted plant growth. Application of different
biocontrol agents such as P. fluorescens, P. lilacinum and P. guilliermondii has been also found
to strengthen the growth of plants via production of natural growth hormones and supplying
many nutritional elements, induction of systemic resistance in plants and lethal effect on
nematodes (Hashem & Abo-Elyousr, 2011).
Plant growth promotion by Paenibacillus spp. and other PGPR is not always the case due
factors like cultivar specificity hence failure of productive association, optimum inoculation
density and excess nutrient at the site (Lebuhn et al., 1997). This may result in inconsistency in
results between experiments. The study by Timmusk et al. (2003) reported 30% reduction of
plant growth and stunted root system on A. thaliana inoculated with P. polymyxa in the absence
of biotic or abiotic stress. Under these conditions, Paenibacillus spp. can be considered as a
deleterious rhizobacterium. But in abiotic and biotic stress P. polymyxa can induce drought
tolerance and antagonizes pathogens (Timmusk & Wagner, 1999).
The conditions under which Paenibacillus spp. application is done should be taken into
consideration to ensure that no deleterious effects occur. The induction of drought tolerance can
be interesting especially in field conditions in places where water is the limiting factor. PGPR
being isolated from natural environments (Lal & Tabacchioni, 2009) can be an advantage for
easier plant colonization and persistence in the field.
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Bakengesa J & Wesemael W
Galling Index was similar for all plants in this study. But higher reduction of galling and
nematode multiplication due to PGPR has been reported in several studies (Khan et al., 2008;
Park et al., 2014). This may be due to factors like production of antibiotic and toxic metabolites
which enhance bio-controlling effects on phytopathogenic microorganisms (Laslo et al., 2012).
The PGPR can also be responsible for induction of plants endogenous defense
mechanisms (M'piga et al., 1997). The activities of enzymes of phenylpropanoid metabolism and
antioxidant have been found to be induced by strain CF05 of P. polymyxa (Mei et al., 2014).
This shows the potential of PGPR on nematode multiplication reduction hence lessen final
population density.
10% BS resulted a higher Pf reduction (p-value>0.05) of M. enterolobii, M. javanica, M.
incognita, M. hapla, M. fallax and M. chitwoodi than 100% BS treatment and control. The study
by Khan et al. (2008) also concluded that ―potted soil treated with 10% concentrations of BS at
the rate of 10 ml/plant at 2 days before nematode inoculation is the most appropriate because it
provided a reasonable level of protection against M. incognita without any phytotoxic effect‖. A
higher Pf on tomato plants treated with 100% than 10% BS may be related to the improved plant
growth, hence nematodes had a better environment. Significant Pf reduction seems to depend on
concentration and inoculum amount and this should be further examined. Optimum dosage and
time for colonization are crucial elements for the success of any bio-control agent.
The interpretation of the results can predict the potential of Paenibacillus spp. for its use
especially in greenhouse production and organic farming where nutrients and specific plant
pathogens are the limiting factor. Small scale production also facilitates application methods.
This can be later extended to field conditions with several biotic and abiotic stresses.
Most studies have been reported on species like M. incognita, M. javanica, and M. hapla.
This study shows the potential of Paenibacillus spp. on other root-knot species like M. fallax, M.
chitwoodi and M. enterolobii. Antagonism of Paenibacillus spp. is not limited to root-knot
nematodes only but also to other plant pathogens like fungi F. oxyporum (Son et al., 2009; Mei
et al., 2014).
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Bakengesa J & Wesemael W
CONCLUSION AND RECOMMENDATIONS
Tested Paenibacillus spp. showed nematistatic and nematicidal effects. Some root-knot
nematode J2s were able to recover in distilled water after exposure into Paenibacillus BS, while
hatching from eggs was completely inhibited with 100% BS. This suggests that its mode of
action for the biological control of root-knot nematodes may be paralysis and antibiosis which
caused mortality, immobility, prevention of invasion and inhibition of hatching. Improvement of
plant growth is also an interesting feature for sustainable production although this may favor
plant pathogens as well. Paenibacillus spp. have potential to reduce deleterious effects of rootknot nematodes hence seem promising as a biological control agent for root-knot nematodes.
For its effective use as a successfully bio-control agent further studies are required on
optimal dosage and inoculum amount. Application timing in order to ensure colonization,
biofilms formation and persistence for effective control should also be figured out. This can
differ between plant species. Increase in number of replicates may also result into statistical
significant results.
ACKNOWLEDGEMENTS
I’m deeply indebted to Almighty God for the grace, wisdom and good health granted to
me throughout this study.
I would like to thank the Belgium Government to fund my study through Vliruos
Scholarship. My very earnest and profound gratitude goes to my promoter Prof. Dr. Wim
Wesemael for his guidance, constructive comments and critical review despite his busy schedule.
Special thanks to the Institute for Agricultural and Fisheries Research (ILVO) for the facilitation
of the research activities and to the staff of ILVO for polite environment at the Institute. A word
of appreciation goes to Prof. Dr. Nicole Viaene, Nancy de Sutter and Steve Baeyen for close
follow ups and directions to make sure things were all right. I am grateful to Nematology family;
Professors, tutors and administration unit for making the program valuable and interesting. Very
sincere greetings are conveyed to my friends and Nematology classmates for their support and
assistance throughout the study.
At last, I would like to dedicate my thesis to my parents, Jasson Bakengesa and Sixta
Imelda Nagabona for their never-ending support and love.
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Bakengesa J & Wesemael W
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